1. Field of the Invention
The present invention relates to the fields of molecular biology, virology and immunology. In general, the present invention discloses construction of replication-deficient viruses belonging to the Flaviviridae family and their use as vaccine in prevention of diseases caused by viruses belonging to this family. More specifically, the present invention provides replication-deficient flaviviruses or pseudoinfectious flaviviruses (PIV aka RepliVAX) and discloses its use as preventive vaccines against flavivirus-associated diseases.
2. Description of the Related Art
The Flavivirus genus of the Flaviviridae family contains a variety of important human and animal pathogens and have been classified into four distinct antigenic complexes based on differences in reactivity in immunological tests. Generally, the flaviviruses circulate between avian or mammalian amplifying hosts and mosquito or tick vectors.
The flavivirus genome is a single-stranded capped RNA of positive polarity lacking a 3′ terminal poly(A) sequence. It encodes a single polypeptide that is co- and post-translationally processed into viral structural proteins, C, prM/M, and E, forming viral particles, and the nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, required for replication of viral genome and its packaging into infectious virions (Chambers, 1990). Virions contain a single copy of viral genomic RNA packaged into a C protein-containing nucleocapsid, surrounded by lipid envelope holding heterodimers of M and E proteins. In contrast to many other enveloped viruses, interaction between nucleocapsid and envelope spikes is not very specific and M/E-containing envelope can efficiently form around nucleocapsid derived from heterologous flavivirus, demonstrating limited level of homology in capsid sequence (Lorenz, 2002). Alternatively, expression of prM and E in the absence of C can lead to formation of subviral particles (SVPs), containing no RNA or C protein (Mason, 1991).
PrM/M-E cassettes producing subviral particles have been the basis of several vaccine candidates that are known in the art. These vaccine candidates include subunit (Konishi, 1992; 2001; 2002; Qiao, 2004), DNA (Phillpotts, 1996; Kochel, 1997; Schmaljohn, 1997; Colombage, 1998; Aberle, 1999; Konishi, 2000; Konishi, 2000; Kochel, 2000; Davis, 2001), and live-vectored (Mason, 1991; Konishi, 1992; Pincus, 1992; Fonseca, 1994; Pugachev, 1995; Colombage, 1998; Kanesa, 2000; Minke, 2004) vaccines. However, these vaccines have serious disadvantages. For instance, the subunit vaccines are safe to use but difficult to produce large quantities; the DNA vaccines are poorly immunogenic, and the viral vectored vaccines suffer from lack of potency in the presence of vector immunity.
Therefore, in spite of a great concern about flavivirus-associated diseases and continuing spread of the flaviviruses into the new areas, antiviral therapeutics have not yet been developed for these infections, and a very limited number of approved vaccines have been produced to-date. Inactivated viral vaccines (INVs) have been licensed to prevent tick-borne encephalitis (TBEV) and Japanese encephalitis (JEV). However, like other inactivated viral vaccines, these vaccines have low limited potency and require multiple vaccinations. Despite these drawbacks the Japanese encephalitis and tick-borne encephalitis INVs have an advantage of good safety records. The only licensed live-attenuated vaccine (LAV) for a flavivirus is the widely utilized live-attenuated vaccine based on the yellow fever virus (YFV) 17D strain that was developed by serial passaging of the wild type Asibi strain of yellow fever virus in chicken embryo tissues. Although this live-attenuated vaccine is considered very safe and effective, cases of yellow fever in vaccinees have been reported, including a recent case in a US military recruit (Gerasimon, 2005). Furthermore, this vaccine is not recommended for use in infants, pregnant women or the immunocompromised individuals due to adverse effects, including vaccine-associated encephalitis.
However, the development of the reverse genetics systems for flaviviruses has led to the production and designing of new types of live-attenuated vaccine, based on rational attenuation of these viruses. This new class of vaccines includes yellow fever virus 17D-based chimeras, in which the yellow fever virus prM-E-encoding genome fragment cassette has been replaced with the prM-E-cassette derived from heterologous flaviviruses (Chambers, 1999). Similar chimeric virus-based approach was applied for dengue- and TBE-based backbones (Pletnev, 2002; Huang, 2003). In most cases, chimeric flaviviruses demonstrate a highly attenuated phenotype and are capable of eliciting efficient protective immune response and protect against following infection with viruses, whose structural proteins are expressed by the chimeras (Monath, 2002). Effective vaccination with these chimeric vaccine candidates appears not to be prevented by pre-existing “vector” immunity (Monath, 2002), which has interfered with potency of recombinant viral vaccines based on other viral vectors. Further, although chimeric flaviviruses might provide a reasonably universal approach to producing new vaccines, there are concerns that the chimeras themselves might be pathogenic (Chambers, 1999) at least in the immunocompromised individuals, or that pathogenic chimeras might arise, since mutations have been detected during the process of propagation of these viruses (Pugachev, 2004).
Another promising direction in vaccine development is based on creating of irreparable deletions in flavivirus genome that make productive virus replication in the vaccinated host either a less efficient or an impossible event. In the latter case, viral genomes encoding the entire replicative machinery, but lacking, for instance, the C-coding region, can be delivered for in vivo immunization either as in vitro-synthesized RNA, capable of self-replication (Kofler, 2004; Aberle, 2005), or, probably, in DNA form (under control of the RNA polymerase II promoters or as in vitro-synthesized RNA, capable of self-replication (Kofler, 2004; Aberle, 2005). Direct immunization with in vitro synthesized defective RNA genomes, which specifies the production of SVPs in the absence of a complete viral replication cycle, has been demonstrated to be safe and effective in producing protective immunity (Kofler, 2004; Aberle, 2005). However, there may be significant obstacles in producing an RNA-based vaccine candidate, due to synthesis, stability, and delivery issues.
Thus, prior art is deficient is deficient in a safe, potent and effective type of vaccine that can be used against diseases caused by infection with viruses belonging to the Flaviviridae family. The present invention fulfills this long-standing need and desire in the art.